1. Field of the Invention
This invention relates to an enzyme electrode for electrochemically determining a particular chemical substance in a solution via an enzyme reaction as well as a biosensor and a measuring apparatus therewith.
2. Description of the Related Art
A process employing an enzyme reaction in combination with an electrochemical reaction has been extensively used for determining a variety of components in, for example, a biological sample. For instance, there has been commonly used a biosensor in which by the catalytic action of an enzyme, a chemical compound in a solution is converted into hydrogen peroxide, which is then determined via an oxidation-reduction reaction. For example, a glucose biosensor can quantify an amount of glucose in a sample by oxidizing glucose with glucose oxidase (GOX) into gluconolactone and hydrogen peroxide, and determining the amount of the hydrogen peroxide because it is proportional to the glucose concentration.
Such a type of sensor generally has a layer restricting permeation of a target chemical compound (hereinafter, referred to as a "permeation restricting layer") as the outermost layer in its electrode. FIG. 9 shows an enzyme electrode having such a structure where an electrode 2 as a working electrode is formed on an insulating substrate 1, on which there are sequentially formed a binding layer 3, an immobilized enzyme layer 4 immobilizing a catalytic enzyme in an organic polymer and a permeation restricting layer 5. Such a permeation restricting layer may restrict an excessive diffusion of the target compound to make an upper limit in determination higher to some extent. In addition, it can prevent the immobilized enzyme layer from being in direct contact with a sample such as urine and blood which may cause deterioration in performance due to protein adhesion or decomposition of the enzyme. A permeation restricting layer has been made of, for example, polyalkylsiloxane (JP-A 10-26601) or silicone (JP-A 6-242068).
JP-A 59-22620 has disclosed a biosensor comprising a permeation restricting layer having a different structure from the above in which a porous TEFLON.RTM. (tetrafluoroethylene) or polyfluorovinylidene film as a permeation restricting layer is placed, covering an electrode.
U.S. Pat. No. 5,696,314 has disclosed an enzyme electrode in which a porous permeation restricting layer including TEFLON particles is formed on an immobilized enzyme layer. In the enzyme electrode, as shown in FIG. 11, an electrode 31 made of, for example, platinum and then an immobilized enzyme layer 32 are formed on a substrate 30. Then, a polymer layer 34 comprising the same enzyme as that in the immobilized enzyme layer 32 is formed via an adhesion layer 33, on which there are sequentially formed a permeation restricting layer 35, an adhesion layer 36 and a protection layer 37.
The U.S. patent has disclosed a porous permeation restricting layer 35 essentially comprising polymer particles, metal particles and polymer binder where the polymer particles and the polymer binder are made of TEFLON. The permeation-restriction layer 35 is formed by screen printing. Specifically, TEFLON binder is dissolved in a fluorine-containing solvent, particles such as alumina and TEFLON particles are added, and then the mixture is roll-milled into an ink. The prepared ink is stenciled to form the permeation restricting layer 35.
The above prior art, however, has the following problems.
There will be described problems in the case of using polyalkylsiloxane or silicone as a permeation restricting layer material. Such a material may cause a problem of insufficient durability for long-term use, which is due to inadequate strength of the permeation restricting layer. An enzyme electrode has a structure of plies made of organic materials such as an immobilized enzyme film which may be swollen in a solution. Therefore, the permeation restricting layer with inadequate strength may be intolerant to such film swelling, resulting in, for example, cracks. Thus, long-term use may cause failure of the enzyme electrode.
When determination is repeated with a sample containing a higher level of contaminant for a long time, a sensor output may be remarkably reduced. It may be caused by deterioration of the original permeation restricting property due to adhesion of the contaminant to the permeation restricting layer. In particular, a body fluid may remarkably deteriorate the permeation restricting property because various materials such as urea compounds in addition to proteins are adhered to the permeation restricting layer.
Furthermore, response may become slower when extending a concentration range to be measured to a higher level because when a sample containing a higher level of target compound is analyzed using a conventional permeation restricting layer, it is inevitable to increase the film thickness due to its limitation in selective permeability, leading to a longer time for stabilizing a diffusion rate within the permeation restricting layer.
A technique using a TEFLON or polyfluorovinylidene (JP-A 59-22620) has the following problem.
A technique using a filter comprising, for example, TEFLON has been conventionally used, where the filter is usually disposed outside of an enzyme electrode, covering the electrode because the fluorine compound is, as apparent from its molecular structure, less adhesive to other organic polymer layers such as an immobilized enzyme layer and thus the filter cannot be formed together with the layers including the immobilized enzyme layer. JP-A 59-22620 has disclosed only a configuration where a film consisting of the above fluorine compound is formed on the tip of the enzyme electrode, but not a configuration where the film is adhesively formed in the electrode surface.
Thus, the above prior art has problems that there is formed a certain gap between the permeation restricting layer and the electrode surface, resulting that 1) response becomes slower due to a longer time for a target compound to reach the electrode surface and 2) it takes a longer time for washing, which leads to a longer waiting time for the next determination.
A permeation restricting layer comprising the above fluorine compound must have pores with a diameter of 10 to 100 .mu.m and be thick adequately to be permeation-restrictive. Thus, response becomes slower and it takes a longer time for washing, leading to a longer waiting time for the next determination.
Furthermore, a permeation restricting layer comprising the above fluorine compound is less flexible. Therefore, the structure is readily broken when a layer disposed nearer to the electrode than the permeation restricting layer is swollen. In particular, the problem is significant when the permeation restricting layer is adjacent to an expansive immobilized enzyme layer.
On the other hand, U.S. Pat. No. 5,696,314 has disclosed a configuration where a permeation restricting layer comprising TEFLON particles and TEFLON binder is formed on an electrode as one part.
As described above, a permeation restricting layer of a polymer with a higher fluorine content such as TEFLON is less adhesive to an adjacent polymer layer such as an immobilized enzyme layer. Therefore, even when the permeation restricting layer is formed with, for example, an immobilized enzyme layer as one part, adhesive strength is insufficient in an interface between these layers. Furthermore, since a permeation restricting layer comprising TEFLON is less flexible, it cannot follow a swollen adjacent layer. Thus, there may readily occur detachment between the permeation restricting layer and its adjacent layer such as an immobilized enzyme layer during operation. Once detachment occurs, there is formed a certain gap between the permeation restricting layer and the electrode surface, resulting that 1) response becomes slower due to a longer time for a target compound to reach the electrode surface and 2) it takes a longer time for washing, which leads to a longer waiting time for the next determination.
When using TEFLON as described in the above publication, it is difficult to prepare a solution due to its less solubility to a solvent. It is, therefore, difficult to deposit a layer by a general process such as spin coating and thus to make the permeation restricting layer thinner. Furthermore, a permeation restricting layer comprising the above fluorine compound must be porous for its permeation restricting property, and therefore, must be thick to some extent. According to the U.S. patent, the thickness is preferably 10 to 40 .mu.m. Thus, it is inevitable to make the permeation restricting layer thick, leading to slower response and a longer washing time.
In addition, a permeation restricting layer comprising TEFLON is less flexible as described above, and therefore tends to be broken when an adjacent layer is swollen. The problem should be also improved. The problem is particularly significant when the permeation-restriction layer is adjacent to an expansive immobilized enzyme layer.
Another technique of the prior art using a fluorine compound will be described, which is not related to an application as a component for a permeation restricting layer.
A fluorine-compound film (TEFLON film) 10 to 50 .mu.m of thickness has been commonly used as an oxygen permeable film and described in, for example, JP-A 56-73342. The film is, however, generally disposed between an immobilized enzyme layer and an electrode, but not on the immobilized enzyme layer. It, therefore, does not act as a permeation restricting layer.
It is also well-known that a NAFION.RTM. film, an ion- exchange film, is disposed on an immobilized enzyme layer, which has been disclosed in, for example, JP-A 8-50112. NAFION is a cation-exchange polymer in which perfluoroalkylene ether side chains having a terminal sulfonic group are attached to a perfluoromethylene principal chain (Formula 1). ##STR1##
A NAFION film disposed on the immobilized enzyme layer may minimize back-diffusion of hydrogen peroxide, reduce variation with time of response to glucose after reaching a peak value and improve response properties. The film is, however, not adequately effective as a permeation restricting layer due to its terminal sulfonic groups. An ion-exchange film is used for preventing permeation of ionic interferent materials interfering an electrode reaction, but little restricts permeation of, for example, excessive glucose.
For a biosensor, it is important to eliminate effects of interferent materials or contaminants. An interferent material refers to a chemical substance which may affect the above oxidation-reduction reaction system to give a positive error in a measurement result, such as ascorbic acid and acetaminophen. A contaminant refers to a chemical substance which may be adsorbed by an electrode surface to give a negative error in a measurement result. For example, Bioindustry, Vol.9, No.12, pp.20-25 (1992) has list albumin, urea, urea compounds and creatinine as a contaminant to a sensor output, i.e., a substance giving a negative error.
JP-A 8-180286 has disclosed a biosensor in which a permeation restricting layer of polyalkylsiloxane and NAFION or acetylcellulose is deposited as an enzyme electrode for eliminating effects of a higher level of interferent materials on a sensor output. FIG. 26 shows its configuration where an electrode 12 is formed on an insulating substrate 11, on which there are sequentially formed a y-aminopropyl-triethoxysilane film 13, an acetylcellulose film 14, a perfluorocarbonsulfonic acid film 15, an immobilized enzyme layer 16 and a polyalkylsiloxane film 17. The publication describes that such a layered structure may prevent a higher level of interferent materials from reaching an electrode surface.
JP-A 3-72254 has disclosed a biosensor in which a permeation restricting layer of NAFION and polyurethane is deposited as an enzyme electrode for eliminating effects of interferent materials or contaminants on a sensor output. FIG. 27 shows its configuration where a working electrode 23, a control electrode 24 and an insulative protection film 25 are formed on a plastic film 22 and an immobilized enzyme layer 29 is formed covering these films. The immobilized enzyme layer 29 consists of a NAFION film 29a, an enzyme layer 29b and a polyurethane layer 29c. The publication describes that such a structure of electrode may reduce permeation or adhesion of an interferent or concomitant material to an electrode.
It has been difficult to eliminate a contaminant such as urea compounds during measurement for a body fluid such as urine or blood containing the contaminant at a higher level when such an enzyme electrode is used. For example, an acetylcellulose film can restrict permeation of a higher molecular-weight compound, but not adequately restrict permeation of a lower molecular-weight compound such as urea.
Therefore, a contaminant such as urea compounds may reach an electrode surface to be irreversibly adsorbed. Thus, repeated use may cause reduction in a sensor output with time, and resultantly such a sensor is less reproductive for repeated measurement or less stable for a long-term use. Furthermore, an urea compound, once adsorbed by an electrode, cannot be easily removed by washing with water. It may lead to a longer waiting time for the next measurement and particularly for repeated measurement, an accumulated negative error may be more significant depending on the measurement number.
Such a problem of adsorption of a contaminant such as an urea compound is significant particularly when platinum is used as an electrode material because an urea compound tends to adhere to platinum. However, an electrode is generally made of platinum having good chemical resistance and good detection properties for hydrogen peroxide. Thus, it has been strongly desired to develop an enzyme electrode which may solve the above problems.